The present invention relates to methods of reducing, growth-inhibiting or eliminating a cell population of a subject in need thereof, to therapeutic cell preparations for practicing such methods and to methods of obtaining such cell preparations. More particularly, the present invention relates to methods of using combined localized cell ablation, such as cryoablation, and vaccination with antigen-presenting cells (APCs), such as dendritic cells, for treating a patient having a disease, such as a metastatic cancer, whose pathology involves a pathological cell population which is characterized by at least one antigen. The present invention further particularly relates to therapeutic cell preparations which comprise immunogenic aggregates of antigen-bearing cells and antigen-presenting cells which are capable of inducing in a subject in need thereof an immune response against a pathological cell population, such as a metastatic cancer cell population, which is specifically associated with at least one antigen.
Malignancies, such as lung and skin cancer, represent a large group of highly debilitating and/or lethal diseases constituting a primary cause of death, and an enormous social and economic burden in the Western world. In particular, cancer now constitutes the leading cause of death in the U.S. of people under the age of 85. The primary cause of lethality of malignant diseases such as lung and skin cancer arises from metastatic spread. In many cases, it is not possible to prevent the onset of metastatic disease since cancers are often metastatic by the time of diagnosis, and even in cases where cancers are diagnosed prior to this stage, complete surgical removal or destruction of primary lesion tissues which are capable of eventually generating metastases may not be feasible. Metastatic disease may be impossible to diagnose at early stages due to the small size of metastatic lesions, and/or the absence of reliable markers in primary lesions upon which to reliably predict their existence. Such lesions may be difficult/impossible to treat via ablative methods due to their being inaccessible, disseminated, and/or poorly localized. Chemotherapy/radiotherapy, the current methods of choice for treatment of certain metastatic malignancies are often ineffective or suboptimally effective, and have the significant disadvantage of being associated with particularly harmful and/or potentially lethal side-effects.
Thus, there exists a longstanding and urgent need for more effective, safer and less invasive methods of treating cancer.
Localized tumor ablation methods, such as cryoablation (i.e. tissue destruction by freeze-thawing), which can be catheter-based, are optimally effective, and minimally harmful/invasive for tumor treatment relative to chemotherapy/radiotherapy since they can be used to destroy cancerous tissues with optimal selectivity and with minimal harmful local/systemic side-effects. Cryoablation is effectively employed for the management of localized, accessible dermatological tumors, hepatocellular carcinoma, renal and prostate tumors, and hepatic colorectal metastases (Han K R, Belldegrun A S., 2004. BJU Int. 93:14-8; Johnson D B and Nakada S Y., 2003. J Endourol 17:627-32; Adam R. et al., 2004. Surg Clin North Am 84:659-71). Compared to surgical excision, catheter cryoablation, by virtue of being less invasive, results in reduced mortality and morbidity, and of being practicable on outpatients, which dramatically decreases treatment cost. In the case of hepatic colorectal metastases, the use of cryosurgery improves the percentages of resectability (Adam R. et al., 2004. Surg Clin North Am 84:659-71). A comparative study on domestic pigs has shown that cryoablation of renal parenchyma is superior to other necrosis-inducing ablations such as microwave thermoablation, radiofrequency energy and chemoablation by ethanol, hypertonic saline and acetic acid gels, in terms of reproducibility, consistency in size and shape, and the ability to monitor by ultrasound (Rehman J. et al., 2004. J. Endourol. 18:83-104). The mechanisms of cryoablation are multifactorial yet they culminate in necrotic cell death secondary to direct mechanical cellular damage induced by ice crystals; and vascular and endothelial injury with eventual ischaemia [Hoffmann N E, Bischof J C., 2002. Urology 60 (2 Suppl 1):40-9]. Freezing-induced immunostimulatory effects have been hypothesized to contribute to the therapeutic effects of cryosurgery, and although a few animal models and clinical case studies contradictorily describe either inhibition (Ablin R J. et al., 1973. Urology 2:276-9), or promotion (Yamashita T., et al., 1982. Gann. 73:222-8) of metastasis/tumor growth following primary tumor cryoablation, the majority of studies prove that cryoablation has no effect on subsequent tumor/metastasis development [Hoffmann N E, Bischof J C., 2002. Urology 60 (2 Suppl 1):40-9]. The main drawback of cryoablation, therefore, is that apart from its local effect on tumors, it does not elicit a systemic anti-cancer response to preclude metastasis.
Immunotherapeutic cancer treatment methods, such as those involving APC vaccination, have the potential to be optimally effective for treatment of inaccessible, disseminated, microscopic, recurrent and/or poorly localized lesions, such as metastatic lesions. One promising immunotherapy avenue involves the use of professional APCs, such as dendritic cells (DCs), to elicit systemic anti-cancer immunity. Dendritic cells are crucially important in antigen capture, processing and presentation to the effector arm of the immune system. Mature dendritic cells direct T-lymphocyte differentiation into effector or memory cells; induce natural killer (NK) cell activation, and induce B-cell differentiation into antibody-forming cells (Ardavin C. et al., 2004. Immunity 20:17-23). A potential role for dendritic cells in eliciting anti-tumor immunity was highlighted by the observation that increased density of dendritic cells present within a tumor correlates with an improved prognosis, and that migration of dendritic cells from the vicinity of the tumor to the draining lymph nodes is essential for the induction of anti-tumor immunity (Tsujitani S. et al., 1992. Int Surg. 77:238-41). Indeed, the potential of dendritic cell administration as an adjuvant treatment capable of potentiating immune-mediated resistance to cancer is supported by many animal experiments as well as initial human trials (Steinman R M, Dhodapkar M., 2001. Int J Cancer 94:459-73). Intratumoral dendritic cell administration has been shown to be potentially therapeutically useful, for example in the treatment of brain tumors (U.S. Patent Application No. 20040057935 to Yu et al.).
Combined radioablation and intratumoral dendritic cell administration has been successfully employed for treating solid tumors with liver micro-metastasis (Chen Z. et al., J Gene Med. 2004 Dec. 6; [Epub ahead of print]), or for treating melanoma or sarcoma tumors in mice (Teitz-Tennenbaum S. et al., 2003. Cancer Res. 63:8466-75). Similarly combined vincristine chemotherapy and intratumoral dendritic cell administration has been successfully employed for treating fibrosarcoma in mice (Shin J Y. et al., 2003. Histol Histopathol. 18:435-47). Such methods, however, are associated with the inherent disadvantages of radiotherapy and chemotherapy. Thus, while immunotherapies involving APC vaccination holds the promise of enabling effective induction of anticancer immunity, such an approach has failed to achieve satisfactory/optimal treatment of numerous types of cancer in humans. Use of combined radiotherapy or chemotherapy and intratumoral dendritic cell administration for treatment of cancer has also been suggested for treatment of brain tumors (U.S. Patent Application No. 20040057935 to Yu et al.).
Dying cells, particularly necrotic cells, which can be generated using localized tumor ablation methods such as cryoablation, are known to be a potent inducer of maturation of dendritic cells capable of inducing specific immunity against cells which express the same antigens as such dying cells (Gallucci S. et al., 1999. Nat. Med. 5:1249-55). Thus, in view of the capacity of localized tumor ablation methods, such as tumor cryoablation, to treat accessible localized tumors, such as bulky primary tumors, while generating dying cells, and in view of the capacity of APCs, which is potentiated by dying cells, to systemically eradicate inaccessible, disseminated and/or poorly localized lesions, such as metastatic lesions, microscopic lesions or recurrent lesions, a potentially optimal cancer treatment strategy would be is to combine such complementary methods so as to achieve treatment of both local and systemic lesions. Such a strategy has the great advantages of avoiding or minimizing the undesirable side-effects inherent to chemotherapy or radiotherapy, and of being practicable using minimally invasive, and broadly mastered techniques, such as catheter cryoablation, and administration of a cancer subject's own APCs.
Several prior art approaches have been employed or suggested in order to treat cancer using APC vaccination combined with tumor cryoablation.
One approach involves treating tumor cells with high temperature, isolating a lysate from the treated cells, and pulsing dendritic cells with the lysate in an attempt to obtain dendritic cells potentially suitable for therapeutic administration to a subject having such a tumor (PCT Publication No. WO/04 018659 to Goletz et al.).
Another approach involves freeze-thawing murine thymoma cells (Galea-Lauri J. et al., 2004. Cancer Immunol Immunother. 53:963-77), human metastatic renal cell carcinoma cells (Gitlitz B J. et al., 2003. J. Immunother. 26:412-9), or human colorectal cancer cells (Bremers A J. et al., 2000. Int J Cancer 88:956-61), and pulsing dendritic cells with lysate of the freeze-thawed cells in an attempt to obtain dendritic cells potentially suitable for therapeutic administration to a subject having such a thymoma, renal cell carcinoma, or colorectal cancer, respectively.
Still another approach involves freeze-thawing tumor cells, preparing soluble antigen from the freeze-thawed cells, and exposing cytokine-induced killer (CIK) cells to the soluble antigen in the presence of dendritic cells, in an attempt to generate activated anti-tumor CIK cells potentially suitable for therapeutic administration to a subject having such a tumor (Yu J. et al., 2003. Beijing Da Xue Xue Bao. 35:141-2).
An additional approach involves freeze-thawing human monoblastoid tumor cells, exposing dendritic cells to the freeze-thawed tumor cells, and then co-culturing the dendritic cells with T-lymphocytes in an attempt to generate proliferating immunostimulated anti-tumor T-lymphocytes potentially suitable for therapeutic administration to a subject having such a monoblastoid tumor (Rad et al., 2003. Cancer Res. 63:5143-5150).
Yet an additional approach involves genetically modifying human melanoma cells to express viral fusogenic membrane glycoprotein (FMG) so as to generate syncitia of tumor cells, freeze-thawing the tumor cell syncitia, isolating released exosomes therefrom, and exposing dendritic cells to the isolated exosomes in an attempt to achieve tumor antigen loading of the dendritic cells, and hence generation of dendritic cells potentially suitable for therapeutic administration to a subject having such a melanoma (Bateman A R. et al., 2002. Cancer Res. 62:6566-6578).
Still an additional approach involves, boiling or freeze-thawing murine thymoma cells, exposing dendritic cells to the boiled or freeze-thawed cells, and then co-culturing the dendritic cells with T-lymphocytes specific for an antigen specifically expressed by the tumor cells in an attempt to generate activated anti-thymoma T-lymphocytes potentially suitable for therapeutic administration to a subject having such a thymoma (Strome S E. et al., 2002. Cancer Res. 62:1884-9).
A further approach involves freeze-thawing murine fibrosarcoma cells (Cohen P J. et al., 1994. Eur J. Immunol. 24:315-9), or human B-lymphoblastoid leukemia cells (Herr W. et al., 2000. Blood 96:1857-1864), pulsing dendritic cells with lysate of the freeze-thawed cells, and co-culturing the pulsed dendritic cells with T-lymphocytes in an attempt to generate activated anti-tumor T-lymphocytes potentially suitable for therapeutic administration to a subject having such a fibrosarcoma, or B-lymphoblastoid malignancy, respectively.
All of the prior art approaches, however, suffer from significant disadvantages. For example, approaches requiring harvesting of tumor cells and/or immune effector cells; genetic modification of tumor cells; in-vitro thermal treatment of tumor cells, in-vitro preparation of subcellular or molecular components of tumor cells; in-vitro treatment of APCs with tumor cells or subcellular or molecular components thereof; and/or in-vitro treatment of APCs with immune effector cells are excessively complex, cumbersome and/or impractical to perform. Approaches involving attempts to generate in-vitro activated anti-tumor immune cytotoxic cells potentially suitable for therapeutic administration, have clearly and explicitly failed to achieve generation of such activated cells. Approaches involving loading of dendritic cells with exosomes isolated from freeze-thawed tumor cell syncitia in an attempt to achieve tumor antigen loading of the dendritic cells have failed to achieve such loading, and hence have failed to achieve generation of dendritic cells potentially suitable for cancer treatment. Approaches involving administration to a human metastatic renal cell carcinoma patient of dendritic cells pulsed with lysate of freeze-thawed tumor cells of the patient have failed to achieve satisfactory/optimal therapeutic efficacy. Approaches involving co-culturing of T-lymphocytes of a colorectal cancer patient with autologous dendritic cells pulsed with a lysate of freeze-thawed tumor cells of the patient in an attempt to generate immunostimulated T-lymphocytes directed against the tumor cells have not attempted administration of such T-lymphocytes to the patient, and hence have failed to demonstrate any therapeutic efficacy. Similarly, approaches involving co-culturing of T-lymphocytes with autologous dendritic cells pulsed with a lysate of freeze-thawed autologous human transformed B-lymphoblastoid cells in an attempt to generate immunostimulated T-lymphocytes directed against the transformed cells have not attempted administration of such T-lymphocytes to a patient bearing such transformed cells, and hence have also failed to demonstrate any therapeutic efficacy. Approaches involving exposure of tumor cells to high temperatures are disadvantageous in that such temperatures tend to denature polypeptides/peptides and epitopes thereof involved in optimal induction of anti-tumor immunity. As well, in the in-vivo context, high temperature treatment of tissues is suboptimal or undesirable relative to low temperature treatment for various reasons. For example, relative to low temperature treatment, high temperature treatment of tissues may be associated with excessive blood loss, thrombogenic risk, and/or patient discomfort,. Furthermore, for treatment of various conditions, high temperature treatment of tissues is far less easily and/or routinely performed by the medical practitioner than cryoablation which is a widely mastered treatment modality. In certain contexts, cryoablation may be advantageously repeated to treat disease relapse, whereas repeated high temperature treatment is contraindicated.
Thus, all prior art approaches have failed to provide an adequate solution for treating diseases such as cancer using combined APC vaccination and localized cell ablation, such as tumor cryoablation.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of treating diseases such as cancer devoid of the above limitation.